Recombinant staphylokinase derivatives and the preparation and applications thereof

ABSTRACT

The present invention relates to the biotechnology field, more particularly, to novel recombinant staphylokinase (RGD/KGD-Sak) derivatives and the preparation the thereof. The derivatives, have a low polymerizing ability, low immunogenicity and a bifunctionality of thrombolytics and anticoagulant. Based on the trimensional structural analysis of the monomer and dimer of recombinant staphylokinases and their biochemical properties, we designed two novel bifunctional staphylokinase molecular structures. Mutant genes were constructed by PCR site-directed mutagenesis, which were then recombined with a prokaryotic vector and used to transform  E. coli . Engineered strains with a high expression level were selected by screening and propagated by fermentation, followed by disruption of the cells, centrifugation to collect inclusion bodies, renaturation, and purification of RGD/KGD-SAK through a two-step method. After lyophilized, the polymerizing ability and immunogenicity of the products decreased significantly. The derivatives can not only activate fibrinogen to lyse thrombus, but also significantly inhibit the platelet aggregation induced by ADP, suggesting that they have the bifunctionality of thrombolytics and anticoagulant.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This patent application is the national stage of an internationalapplication and claims priority to Chinese patent application, serialnumber CN00111627.4, filed on Jan. 28, 2000, the subject matter of whichis incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to novel recombinant staphylokinasederivatives. More particularly, the invention relates to the recombinantstaphylokinase derivatives with a significant decrease in polymerizingability as compared with wild-type staphylokinase and thebifunctionality of thrombolytics and anticoagulant. The invention alsorelates to the preparation of these staphylokinase derivatives, and tothe application of these recombinant staphylokinase derivatives as athrombolytic drug.

BACKGROUND OF THE INVENTION

The naturally occurring staphylokinase (Sak) is a proteolytic enzymeproduced by the lysogenic phage: of Staphylococcus aureus, and consistsof 136 amino acid residues. Indeed, Sak is not an enzyme in nature, butit forms a 1:1 complex with plasminogen (plg); in human plasma, whichcomplex is then activated into Sak plm by the trace of plasmin (plm) onthe surfaces of blood clots. Sak plm is a potent plasminogen activatorto activate the free form of plg into plm, which in turn catalyzes thedegradation of fibrin, the main matrix of thrombus, thus resulting inthe lysis of thrombus. Sak has fibrin specificity in plg activation andacts more efficiently than other thrombolytic agents to lyse oldthrombus and platelet-rich thrombus. Thus, Sak is an efficient andspecific thrombolytic agent (Collen D et al, Nature Medicine 4, 279-284(1998)). At present, recombinant staphylokinases are produced by severalcompanies in the world, but they differ from each other in genestructures. The thrombolytic therapy of acute myocardial infarction(AMI) with recombinant staphylokinases studied by D. Collen of Belgiumcompleted the clinical trial stage II. ShiXin Centre (Chengdu, China)also finished clinical trial stage I of AMI and the effect was quitegood. In 1994 Shanghai Medical University constructed a Sak gene,accomplished the high level expression in E. coli, ad finished the pilotprocess. They have applied for the permission of clinical trials totreat acute cerebral infarction. However, as a heterologous protein, Sakmay have strong antigenicity when administered to patients. Though nosevere allergic reaction was reported in the clinical trials, Sakinduced a high titer of neutralizing antibodies in most patients twoweeks after administration, arguing against its repeated administration(Declerck P J et al, Thromb Haemost 71, 129-133 (1994)). Moreover, itwas discovered in the study of recombinant staphylokinases thatstaphylokinases tend to form dimers, even polymers. The formation ofpolymers increases its immunogenicity.

During the thrombolytic therapy, thrombolytic drugs, are often combinedwith anti-thrombin or anti-platelet drugs such as heparin and aspirin topromote thrombolysis and to prevent reinfarction. Recent studies ofthrombolytic auxiliary drugs are remarkable. Arg-Gly-Asp (RGD) andLys-Gly-Asp (KGD) are functional sequences against platelet aggregation.They competitively bind to the glycoprotein membrane receptor IIb/IIIaassociated with the platelet membrane aggregation, thus preventing thebinding of fibrinogen to its receptor and blocking the reformation ofthrombus (Frishman W H et al, Am. Heart J. 130, 877-892(1995); Nichols AJ et al, Trends Pharmacol. Sci 13, 413-417(1992)). Introducing theRGD/KGD sequence into the cDNA of a thrombolytic agent e.g. urokinaseunder an appropriate conformational restriction, the product expressedwill have the bifunctionality of thromboytics and anticoagulant (Smith Jet al, J. Biol. Chem. 270, 30486-30490 (1995)). However, it wasindicated in the clinical trials that the thrombolytic effect ofurokinase was significantly lower than staphylokinase (50% versus 75%).Furthermore, various chemical mimics have been developed based on theRGD/KGD sequence such as Tirofiban, Lamifiban, Lefradafiban, Orbofiban,Xemilofiban, Integrinlin and the like, which could block the IIb/IIIareceptor. When administered in combination with thrombolytics, theincidence of reinfarction would be significantly decreased (Frishman W Het al, Am. Heart J. 130, 877-892 (1995); Verstraete M et al, 49, 856-884(1995)).

The object of the invention is to provide novel staphylokinasederivatives, which escape from forming dimer and have thebifunctionality of thrombolytics and anticoagulant, and the preparationthereof.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to novel staphylokinase derivatives, whichescape from forming dimer and have the bifunctionality of thrombolyticsand anticoagulant, and to the preparation and application thereof. Inthe present invention, novel Sak molecular structures were designed withstructural biology and prepared by genetic engineering. Besides theefficient and specific thrombolytic effect, the resulting product havenew properties such as low polymerizing ability and anti-plateletaggregation. The preparing process is simple and safe. The yield, Purityand activity of the products are substantially the same as that ofwild-type Sak.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the SDS-PAGE photograph of various concentrations of thewild-type and the recombinant staphylokinase of the present invention,wherein: lane 1, molecular standard lane 2, RGD-Sak (3 mg/ml); lane 3,RGD-Sak (30 mg/mL) lane 4, Sak (3 mg/ml) and lane 5, Sak (30 mg/ml).

FIG. 2 shows the results of sensitizing test of wild-type staphylokinaseand RGD-Sak on guinea pigs.

FIG. 3 shows the results of anti-platelet-aggregation test of RGD-Sak.

DETAILED DESCRIPTION OF THE INVENTION

Wild-type Sak is an ellipsoid molecule, which comprises an α-helixconsisting of 12 amino acid residues covered by two β-sheets consistingof 5 and 2 β-strands respectively, extending from the amino acid residue20. The 20 NH₂-terminal amino acids are extending outwards flexibly,whose functions can hardy be deduced from the crystal structure. Sakshows obvious asymmetry in hydropathy and the active region is largelyat the hydrophile side (Zhan C H et al, Acta Cryst. D52, 564-565 (1996),Rabijns A et al, Nat. Struct. Biol. 5, (1997)). To determine the bindingregions of the Sak dimer, molecular joining was performed with GRAMMV1.03 software developed by Vakser I A (Rockefeller University, USA) onthe basis of the X-ray diffraction crystal structure of monomeric Sak.Using one Sak molecule as the receptor and another Sak molecule as theligand, the Sak receptor was searched for the binding region withrespect to the Sak ligand. 10 complex structures obtained by joiningwith the global high resolution joining parameters recommended by theauthor were searched. The modelling was performed on a SGI 02 graphicworkstation.

TABLE 1 Global high resolution joining parameters Matching mode(generic/helix) mmode = generic Grid step eta = 1.7 Repulsion(attraction is always −1) ro = 30 Attraction double range (fraction ofsingle fr = 0 range) Potential range type (atom radius, grid step) crang= atom radius Projection (blackwhite, gray) ccti = gray Represention(all, hydrophobic) crep = all Number of matches to output maxm = 10Angle for rotations, deg (10, 12, 15, 18, 20, ai = 10 30, 0-no rot)

The electrostatic potential and hydrophobicity, analysis indicated thatmonomeric Sak was significantly asymmetric in hydropathy. Silence et alshowed by random mutagenesis that amino acids determining the activitymainly situated on the hydrophilic side (Silence K et al, J. Biol. Chem.270, 27192-27198 (1995)). The hydrophobic side of Sak has two mainhydrophobic regions (HR), which lie at residues 47-56(HR1) and104-113(HR2) respectively, wherein HR2 is more hydrophobic. In thestructure mode of the dimer constructed, the interaction between thehydrophobic regions is very important and has two binding manners,HR1-HR2 and HR2-HR2. Because HR1 is close to the active region, theactive region of one Sak molecule will be covered when two Sak moleculesbind to each other in the manner of HR1-HR2, probably retaining theactivity of one Sak molecule only. When they bind to each over in themanner of HR2-HR2, the activity appears not to be influenced largely.

The interface of protein interface on is usually between 600 and 1300Å², and each molecule provides 10-30 contacting residues. However, thereis a so-called “hot spot” in the interface in which only 3-5 amino acidsprovide about 80% of the binding energy. The change of these residueswill result in significant decrease of the binding ability of thecomplex (Li B et al, Science 270, 1657-1660 (1995)). Thus, irrespectiveof the particular binding manner of the dimer, the formation of dimerswill probably be prevented when the main binding residues of the HR2 arechanged. Phe111, to strongly hydrophobic amino acid, situates in thecore region of, HR2 and is far away from active region. In a preferredembodiment of the invention, it is replaced by Asp, a strongly polaramino acid, to disrupt the hydrophobic effect. The mutant was expectedto retain the activity. Further, since peptides of RGD/KGD sequence caninhibit platelet aggregation, and the loop region within the β-sheets isquite free in conformation, Lys109 was changed into Arg in the presentinvention, resulting in a RGD-Sak sequence, or Lys109 was not changed,which results in a KGD/Sak sequence. The amino acid sequence of RGD-Sakof the invention is set forth it SEQ ID NO: 1, and the amino acidsequence of KGD-Sak is set forth in SEQ ID NO: 3.

In another aspect, the present invention relates to a method for theproduction of the staphylokinase derivatives of the present invention,which comprises: preparing a DNA fragment comprising at least the partof the coding sequence of staphylokinase that provides for itsbiological activity; performing in vitro site-directed mutagenesis onthe DNA fragment to replace one or more codons for wild-type amino acidswith a codon or codons for another (other) amino acid(s); cloning themutated DNA fragment into a suitable vector; transforming ortransfecting a suitable host cell with the recombinant vector; culturingthe host cell under conditions suitable for the expression of the DNAfragment; and recovering and purifying the desired staphylokinasederivatives from the culture medium.

The site-directed mutagenesis can be performed by polymerase chainreaction (PCR). In a preferred embodiment a first amplification iscarried out by using a backward-primer and mutating-primer, with plasmidpST-Sak as the template. After recovered and purified by agarose gelelectrophoresis, the double stranded fragment amplified was used as aprimer to carry out a second amplification with a forward-primer usingplasmid pST-Sak as template again. Following purifications the fragmentobtained is used as a template in a third amplification together withthe forward-primer and the backward-primer. The product was blunted withKlenow fragment, EcoRI and BamHI digested, ligated with EcoRI/BamHIdigested pUC19, and transformed into E. coli strain JM109. A positiveclone was selected by screening through digestion analysis, and thepresence of the desired mutation at the expected position was verifiedby nucleotide sequence analysis. The sequencing analysis was performedby Genecore Biotechnology Co on an ABI 377 sequencer. Then, the RGD-Sakgene was removed by EcoRI and BamHI digestion, and ligated into thecorresponding site of expression vector pLY4.

SEQID:5 forward-primer: 5′-CGC GAA TTC ATG TCA AGT TCA TTC GAC-3′SEQID:6 backward-primer: 5′-CGC GGA TCC TTA TTT CTT TTC-3′ SEQID:7mutating-primer(I): 5′-TAA ATC TGG GAC GAC GTC ACC ACG TTC TGT TATAGG-3′(a PstI site introduced, used to construct a RGD-Sak gene) SEQID:8mutating-primer(II): 5′-ATC TGG GAC GAC GTC ACC TTT TTC TG-3′ (a PstIsite introduced, used to construct a KGD- Sak gene)

Different DNA fragments comprising the nucleotide sequences coding forthe staphylokinase derivatives of the present invention can be preparedfollowing the above method. These fragments comprise the nucleotidesequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

Recombinant expression plasmids were obtained by ligating the DNAfragments of the invention to an expression vector. The invention is notlimited to any particular expression vector provided that it can berecombined with the above DNA fragment yielding plasmids suitable forexpression. In a preferred embodiment of the invention, a prokaryoticexpression vector is used for example pLY-4, T7 expression system, PLexpression system and the like.

The above recombinant expression vector may be introduced into asuitable host cell by conventional procedures. The invention is notlimited to any particular host cells, provided that they can express therecombinant expression vectors. In a preferred embodiment of theinvention, an E. coli strain, is used, for example K802, JF1125, JMseries, DH5α, and the likes.

The expression product of the invention was present as inclusion bodiesin the engineered cells. The desired product can be isolated andpurified from the inclusion bodies by conventional procedures, forexample disrupting the cells by a French press and collecting theinclusion bodies by centrifugation.

For all basic manipulations of the molecular biology in aboveembodiments, see Molecular Cloning, A laboratory Manual.

Furthermore, the nucleic acids and the corresponding polypeptides of theinvention include the sequences that are different from the sequencesset forth in SEQ ID NO: 1-4 due to silent mutations. These sequencemodifications include for example nucleotide substitutions that do notalter the amino acid sequence (for example, different codons for thesame amino acid or degenerate sequences). The amino acid sequences ofthe homologous polypeptides may differ from that of SEQ ID NO: 1 or SEQID NO: 3 in that one or more amino acid residues are inserted deleted orreplaced with other different amino acid residues. Preferably, the aminoacid changes are of minor nature, i.e. conservative amino acidsubstitutions which will not influence the folding and/or activity ofthe protein significantly; small deletions, usually of 1 to about 30amino acids in length, small extensions at amino or carboxyl terminus,e.g. the methionine residue at amino terminal; small connective peptideup to about 20-25 residues in length; or small extensions that willfacilitate purification by changing the net charge or that have otherfunctions, e.g. poly-histidine tract, antigenic epitopes or bindingdomains.

The present inventor discovered that the thrombolytic and anticoagulantfunctions of the expression products remained almost unchanged when thecoding sequence of the 6-10 amino acids NH₂-terminal of RGD-Sak orKGD-Sak was removed by deletion mutation; the thrombolytic activity ofthe expressions was lost when the coding sequence of the 10-15NH₂-terminal amino acids was removed; and the above bifunctionality ofthe expression products remained almost unchanged when the coding,sequence of the 15 NH₂-terminal amino acids was removed and the Ser atposition 16 was changed to Lys.

EXAMPLE 1 Design, Preparation and Characterization of RGD-Sak

a. The Identification of Wild-Type r-Sak

Wild-type r-Sak, prepared by our laboratory (970923) was more than 98%pure and stored at −70° C.

Reductive and non-reductive SDS-PAGE performed according to the methodof Laemmli (see Molecular Cloning, A Laboratory Manual).

Loading buffer: containing 0.0625 mol/L Tris-HCl pH6.7, 2% SDS, 10%glycerol, 5% mercaptoethanol and 0.001% bromophenol blue.

Sample treating and loading a vial of lyophilized sample (3 mg/vial,stored at −70° C. for more tan 3 months) was dissolved in 3 ml ddH₂O.The loading volume was 10 μl.

Gel staining: Coomassie brilliant blue R-250 or silver staining

Scanning the protein bands in the gel: scanning with ImageMaster® VDS(Pharmacia) and analyzing the amount of protein contained in each bandswith appended software.

After electrophoresis, the gel is stained with Coormassie brilliantblue, and dense bands appeared at positions corresponding to relativemolecular weights of about 15.5 kD, 31 kD, 46 kD and 62 kD.

Determining the activities by inverted casein gel plate method the abovegel was sequentially washed with 2.5% Triton X-100 solution anddistilled water thoroughly, placed on the agar gel plate (comprising 1%agar) containing fibrinogen, human plasminogen and thrombin, andincubated at 37° C. for 8 hours. Clear lysis bands appeared at positionscorresponding to the above molecular weights, suggesting that wild-typer-Sak tends to form anti-SDS polymers during the storage of wild-typer-Sak, which is stable and active.

b. The Molecular Simulation of the Staphylokinase Dimer and ReasonableDesign of Mutants

The modeling work was performed on a SGI 02 graphic workstation withGRAMM V1.03, a molecule joining software developed by I. A. Vakser(Rockefeller University, USA).

To determine the binding region of Sak dimer, Sak-to-Sak joining wasmade with GRAMM V1.03 on the basis of the X-ray diffraction crystalstructure of monomeric Sak.

Phe111 was replaced with Asp, a strongly polar amino acid in theinvention to disrupt the hydrophobic interaction. The mutant wasexpected to retain the activity. Further, since peptides of RGD sequencecan inhibit platelet aggregation and the loop region)), within theβ-sheets is quite free in conformation Lys109 was also changed to Arg toyield RGD sequence.

c. The Cloning of RGD-Sak Gene and the Construction of ProkaryoticExpression Plasmid

Using pST-Sak as template, a first amplification was carried out withthe forward-primer and mutating-primer (II) shown below. After the 351bp fragment amplified was recovered from agarose gel and purified, itwas used to carry out a second amplification with the backward-primershown below, using pST-Sak as template again. Following purification,using the 408 bp fragment as template a third amplification was carriedout with the forward-primer and the backward-primer. The product wasblunted with Klenow fragment EcoRI and BamHI digested, ligated to pUC19,and transformed. A positive clone was selected by digestion analysis,and the presence of the desired mutations was verified by nucleotidesequence analysis. The sequence analysis was performed by GenecoreBiotechnology Co. on an ABI 377 sequencer. Then, the KGD-Sak gene wasremoved by EcoRI and BamHI digestion, and ligated into the correspondingsite of the expression vector pLY-4.

SEQID:5 forward-primer: 5′-CGC GAA TTC ATG TCA AGT TCA TTC GAC-3′SEQID:6 backward-primer: 5′-CGC GGA TCC TTA TTT CTT TTC-3′ SEQID:8mutating-primer(I): 5′-ATC TGG GAC GAC GTC ACC TTT TTC TC- 3′(a PstIsite introduced)

All nucleic acid modifying enzymes were purchased from GIBCO BRL andPromega. Oligonucleotides were synthesized by DNA Synthesis Group ofJohns Hopkins University (USA).

E. coli strain JM109 and pUC19 were kept by our laboratory E. colistrain JF1125 and prokaryotic expression vector pLY-4 were kindlyprovided by Prof. Xin-Huan Liu of the Institute of Biochemistry of theChinese Academe of Science (China). pST-Sak was constructed by ourlaboratory (Chinese Patent NO. 94 1 12105.4).

The gene of interest was ligated into pLY-4 aid transformed into E. colistrain JF1125. The plasmid was prepared and identified by correspondingdigestion analysis. The characteristic fragment was obtained, verifyingthe positive clone.

The E. coli strain JF1125 transformed with pLY-4 RGD-Sak was cultured inM9CA culture medium at 30° C. until OD600 reached 0.6. Then thetemperature was increased to 42° C. and the culturing was continued foranother 3 hours to induce expression. The product expressed was analyzedby SDS-PAGE. After the electrophoresis, one half was stained byCoomassie brilliant blue. A dense band was observed at a molecularweight of about 15.5 kD in the lane of the lysate of induced bacterialcells, which accounted for more than 50% of the total proteins of thebacterial cells as judged by scanning. The other half was placed on acasein gel plate after SDS was removed, and incubated at 37° C. forseveral hours. There was a clear region corresponding to 15.5 kD. Inother words, casein at this position was degraded, suggesting thatRGD-Sak had a fibrolytic activity. After the cells were crushed andcentrifuged, it was discovered that the 15.5 kD band was mainly presentin the pellet, while it could hardly be observed in the supernatantindicating that the product expressed exists as inclusion bodies

d. Inducible Expression in Engineered Strains

The engineered strains were screened for high lever of expression (e.g.the recombinant protein expressed accounted for more than 50% of thetotal protein of the cell). Low density fermentation was carried outwith the strain selected in a 10 L fermentor. After 3 hours oftemperature induction culturing, cells were spun down, washed in PBS,and stored at −70° C. until use 80 g wet cells were obtained from a 10 Lculture. The wet cells were suspended in PB buffer, disrupted by a highpressure homogenizer and centrifuged. Samples were taken for SDS-PAGE.The results indicated that the protein of interest was present in thelane of the pellet with a band stained densely at the position of amolecular weight of 15.5 kD, and that hardly any stain could be observedat the corresponding position of the supernatant, suggesting thatRGD-Sak mainly exists as inclusion bodies.

e. Isolation, Solubilization and Renaturation of Inclusion Bodies

After disrupting by pressing, 80 g cells of the engineered strain werecentrifuged at 10,000 rpm and 20 g inclusion bodies was obtained. Afterthe inclusion bodies was washed in 0.05 mol/L PB and centrifuged at5,000 rpm, it was dissolved in a solution containing 0.1 mol/L PB pH5.0,6 mol/L, guanidium hydrochloride, 0.5% β-mercaptoethanol, and incubatedat room-temperature until the solution became clear. Afterultracentrifugation at 30,000 rpm the pellet was discarded and thesupernatant was diluted for renaturation in 0.1 mol/L PB pH5.0 and 0.5%β-mercaptoethanol

f. Sephadex G-10 and S-Sepharose FF Column Chromatography

After concentration by ultrafiltration (MW 1000, Millipore) thesupernatant was filtrated through a Sephadex G-10 column. The filtratewas applied to an S-Sepharose FF column equilibrated previously by 10bed volumes of 0.1 mol/L PB buffer pH 5.0. A chromatograph (Waters) wasused to control the flow rate and to detect the protein peak. Afterloading, the column was washed to baseline with PB buffer and elutedwith a 0-1 mol/L gradient of NaCl. The fractions eluted were collected.The distribution of the desired protein was analyzed by SDS-PAGE and theconcentration determined by Bradford method (the reagents used werepurchased from Bio-Rad).

All chromatography operations were of routine work to those skilled inthe art.

g. The Identification of the Purity and the Determination of theMolecular Weight.

The sample was analyzed by SDS-PAGE according to Molecular Cloning, ALaboratory Manual. After stained with Coomassie brilliant blue R-250,the gel was scanned with Pharmacia Imagemaster VDS to determine thepurity and molecular weight of the protein. Consequently, it wasdetermined that the purity was above 95% and that the molecular weightwas about 15.5 kD

h. The Determination of the Biological Activity

Casein gel plaque method (Pipemo A G et al, J. Exp. Med. 48(1), 223-234(1978)) and chromogenic substrate method (Lijnen H R et al, J. Biol.Chem. 266, 11826-11832 (1991)) were carried out to determine thebiological activity. The specific activity was about 90,000-100,000HU/mg. For the definition of the Unit, see Tang Q-Q et al, DrugBiotechnology (Chinese) 4(1), 1-4(1997).

i. The Determination of the Km and Kcat Value of Sak Plasmin Complex andRGD-Sak Plasmin Complex

2 μmol/L Sak or RGD-Sak was incubated with 2 μmol/L plasminogenrespectively in 0.1 mol/L PB pH5.0 at pH7.4 and 37° C. for 30 min toform complexes with plasmin. Then catalytic amount of complex was taken(5 nM) to react for 0-10 min in the following systems in 0.1 M PB atpH7.4 and 37° C., and OD405 was recorded every 30 sec. Eachconcentration of plasminogen was assayed in triplicates and averaged.

Reaction System Final Concentration Sak · plasmin (RGD-Sak · plasmin) 5nmol/L chromogenic substrate 1 mol/L plasminogen 1-30 μmol/L

The activation of plasminogen by RGD-Sak plasmin corresponded to theMichaelis-Menten equation (table 1)

TABLE 1 the comparison of enzymatic kinetic constants of the plasminogenactivation by RGD-Sak · plasmin and Sak · plasmin Km(μmol · L⁻¹)Kcat(s⁻¹) Kcat/Km Sak · plasmin 6.42 1.03 0.16 RGD-Sak · plasmin 12.501.41 0.11j. The Test of Polymerizing Ability

Wild-type Sak was used as a control. The samples were dissolved inphysiological saline. Two protein concentrations, 30 mg/ml (high) and 3mg/ml (low), were tested. The solutions were kept at room temperature.Samples were taken every 24 hr and analyzed by electrophoresis.

At both protein concentrations, the polymerizing ability of RGD-Sak wassignificantly lower than that of wild-type Sak (FIG. 1).

k. The Sensitizing Test on Guinea Pigs

Both recombinant wild-type Sak and mutant RGD-Sak were dissolved insterile physiological saline at a concentration of 2500 U/ml for thesensitizing test. For each administration intact vials were taken toprepare the fresh solutions in a sterile way, 20 healthy guinea pigswere assigned to two groups randomly, with 10 guinea pigs each. Theguinea pigs were i.p. injected with r-Sak or RGD-Sak at a dose of 0.15mg/kg every other day for three times. A first and a second i.v. attackat 0.3 mg/kg were performed on day 14 and 21, respectively. 2 healthyand non-injected: guinea pigs were: i.v. injected with above samples at0.3 mg/kg and observed for other presence of similar response to excludethe pharmacological and pathological interference of the samples.

The group injected with wild-type r-Sak 8 guinea pigs showed a positiveresponse of grade IV and 2 showed, a positive response of grade II.

The group injected with RGD-Sak: 2 guinea pigs showed a positiveresponse of grade I and the others showed no obvious response.

Grade I response: mild cough Grade II response: cough several times,quiver Grade III response: quiver violently Grade IV response:convulsion, spasm, incontinence of the feces and urine, shock to death

The antibody levels in the sera from the guinea pigs, immunized for 1-3weeks were tested by ELISA, wherein wt-Sak and RGD-Sak were used asantigens, respectively. In the fist week, the antibodies against eitherantigen were low. In the second week, the antibody level of the wt-Sakgroup (n=10) increased to 1:800, whereas the antibody lever of theRGD-Sak group (n10) was 1:400. In the third week, the wt-Sak groupincreased to 1:3200, whereas the RGD-Sak group remained at 1:400. Thus,the immunogenicity of the RGD-Sak decreased significantly as comparedwith wt-Sak (FIG. 2B).

The above results indicated that the immunogenicity of the RGD-Sak wasdecreased significantly as compared with wt-Sak.

l. The Platelet Aggregation Inhibitory Assay

Fresh blood anticoagulated with 1/10 volume of 110 mmol/L sodium citratewas centrifuged slowly (150 g, 10 min) to get the platelet-rich plasma(HRP). RGD-Sak was added to HRP to a final concentration of 2 μmol/L andthe mixture was incubated at 37° C. for 2 min with continuous stirring.Then ADP was added to a final concentration of 2 μmol/L as an inducer.The platelet aggregation rate was determined within 5 min with atwo-channel platelet aggregator (CHRONO-LOG560). Wild-type r-Sak. (2μmol/L) and physiological saline were assayed as controls. ADP waspurchased from Sigma and other reagents were of analytic grade made inChina.

Consequently, the aggregation rate of the RGD-Sak, group (5%±2%, n=3)was significantly lower than that of the RGD-Sak group (58%±3%, n=3) andthat of the physiological saline group (59%±3%, n=3), suggesting thatRGD-Sak has a powerful potency to inhibit platelet aggregation inducedby ADP (FIG. 3).

m. The Thrombolytic Assay on Animals

The animal thrombolytic assay was performed with RGD-Sak prepared in thepresent invention, verifying that RGD-Sak retained the same thrombolyticproperty as that of wild-type Sak.

(i) Treating experimental rabbit femoral artery thrombosis with RGD-Sak:the treatment group of RGD-Sak, the treatment group of wild-type Sak andthe control group of blank each consisted of 6 animals. It was indicatedby arteriography that the femoral artery under the middle segment wasnot visible before treatment. When photography was repeated 60 minutesafter i.v. injection of 0.1 mg/kg RGD-Sak, the femoral artery was filledthoroughly and the blood cycle was recovered which was consistent withthe wild-type Sak group, while in the control group, the femoral arterydid not appear to be filled.

(ii) Treating experimental rabbit hyphema with RGD-Sak the treatmentgroup of RGD-Sak, the treatment group of wild-type Sak and the controlgroup of blank each consisted of 6 animals, 4 hours after intraocularinjection of 10-20 μg RGD-Sak, it was observed that the hyphema clot waslysed and the red blood cells settled and formed an interface withaqueous humor. The intraocular hematocele was eliminated after 24 hours.This is consistent with the wild-type Sak group. However, the hyphema inthe control group was not significantly changed.

(iii) RGD-Sak thrombolytic therapy was safe and efficient for induced bythe treatment of acute myocardial infarction experimental dog coronaryarterial thrombosis. The experimental group consisting of 6 animals wasgiven RGD-Sak at 0.3 mg/kg body weight by i.v. infusion; and the controlgroup consisting of 6 animals was given physiological saline instead ofRGD-Sak by i.v. infusion Coronary arteriography was carried out beforeand after dosing. Before administration, it was shown that the leftanterior descending branch of the coronary artery was unfilled or filledincompletely in the animals of both groups. Arteriography was performed30 minutes after treatment. It was shown that the left anteriordescending branch was refilled in the animals of the experimental group,and the animals survived. As for the control group, there was nosignificant change in the region filled incompletely and the animalsdied several hours later.

(iv) RGD-Sak thrombolytic therapy was safe and efficient the treatmentof acute cerebral infarction induced by experimental pig intracranialarterbial thrombosis. The experimental group-consisting of 6 pigs wasgiven RGD-Sak at 0.2 mg/kg body weight by i.v. infusion; and the controlgroup consisting of 6 pigs was given physiological saline instead ofRGD-Sak by i.v. infusion. Intracranial DSA arteriography was performedbefore and after administration. Before administration, it was shownthat there were some intracranial arteries filled incompletely in theanimals of both groups. Angiography was performed 30 minutes aftertreatment. It was shown that the intracranial arteries were refilled inthe animals of the experimental group, and the animals survived. As forthe control group, there was no significant change in the intracranialarteries filled incompletely and the animals died several days later.

EXAMPLE 2 Design, Preparation and Characterization of KGD-Sak

a. Identification of Wild-Type r-Sak

Wild-type r-Sak, prepared by our laboratory (970923), was more than 98%pure and stored at −70° C.

Reductive and non-reductive SDS-PAGE; performed according to the methodof Laemmli (see Molecular Cloning, A Laboratory Manual).

Loading buffer: containing 0.0625 mol/L, Tris-HCl pH6.7, 2% SDS, 10%glycerol, 5% mercaptoethanol and 0.001% bromophenol blue.

Sample treating and loading: a vial of lyophilized sample (3 mg/vial,stored at −70° C. for more than 3 months) was dissolved in 3 ml ddH₂O.The loading volume was 10 μl.

Gel staining: Coomassie brilliant blue R-250 or silver straining.

Scanning the protein bands in the gel, scanning with ImageMaster® VDS(Pharmacia) and analyze the amount of protein contained in each bandswith appended software.

After electrophoresis, the gel is stained with Coomassie brilliant blue,and dense bands appeared at positions corresponding to relativemolecular weights of about 15.5 kD, 31 kD, 46 kD and 62 kD.

Determining the activities by inverted casein gel plate method the abovegel was sequentially washed with 2.5% Triton X-100 solution anddistilled water thoroughly, placed on the agar gel plate (comprising 1%agar) containing fibrinogen, human plasminogen and thrombin, andincubated at 37° C. for 8 hours. Clear lysis bands appeared at positionscorresponding to the above molecular weights suggesting that wild-typer-Sak tends to form anti-SDS polymers during the storage of wild-typer-Sak, which is stable and active

b. Molecular Simulation of the Staphylokinase Dimer and ReasonableDesigning of Mutants

The modeling work was performed on a SGI 02 graphic workstation withGRAMM V1.03, a molecule joining software developed by I. A. Vakser(Rockefeller University, USA).

To determine the binding region of dimeric Sak, Sak-to-Sak joining wasmade with GRAMM V1.03 on the basis of the X-ray diffraction crystalstructure of monomeric Sak.

Phe111 was replaced with Asp, a strongly polar amino acid, in theinvention to disrupt the hydrophobic interactions. The mutant wasexpected to retain the activity. Further, singe peptides of KGD sequencecan inhibit platelet aggregation and the loop region within the β-sheetsis quite free in conformation, the thrombolytic activity was notexpected to be affected.

c. Cloning of KGD-Sak Gene and the Construction of ProkaryoticExpression Plasmids

Using pST-Sak as template, a first amplification was carried out withthe forward-primer and mutating-primer(I) shown below. After the 351bpfragment amplified was recovered from agarose gel and purified, it wasused to carry out a second amplification with the backward-primer shownbelow, using pST-Sak as template again Following purification, using the408bp fragment as template, a third amplification was carried out withthe forward-primer and the backward-primer. The product was blunted withKienow fragment, EcoRI and BamHI digested, ligated to pUC 19, andtransformed. A positive clone was selected by digestion analysis, andthe presence of the desired mutations was verified by nucleotidesequence analysis. The sequence analysis was performed by GenecoreBiotechnology Co. on an ABI 377 sequencer. Then, the RGD-Sak gene wasremoved by EcoRI and BamHI digestion, and ligated into the correspondingsite of the expression vector pLY-4.

SEQID:5 forward-primer: 5′-CGC GAA TTC ATG TCA AGT TCA TTC GAC-3′SEQID:6 backword-primer: 5′-CGC GGA TCC TTA TTT CTT TTC-3′ SEQID:7mutating-primer(I): 5′-TAA ATC TGG GAC GAC GTC ACC ACG TTC TGT TATAGG-3′ (a PstI site introduced)

All nucleic acid modifying enzymes were purchased from GIBCO BRL andPromega Oligonucleotides were synthesized by DNA Synthesis Group ofJohns Hopkins University (USA).

E. coli strain JM109 and pUC19 were kept by our laboratory. E. colistrain JF1125 and prokaryotic expression vector pLY-4 were kindlyprovided by Prof. Xin-Huan Liu of the institute of Biochemistry of theChinese Academe of Science (China). pST-Sak was constructed by ourlaboratory (Chinese Patent).

The gene of interest was ligated into pLY-4 and transformed into E. colistrain JF1125. The plasmid was prepared and identified by correspondingdigestion analysis. The characteristic fragment was obtained, verifyingthe positive clone.

The E. coli strain JF1125 transformed with PLY 4 KGD-Sak was cultured inM9CA culture medium at 30° C. until OD600reached 0.6. Then thetemperature was increased to 42° C. and the culturing was continued foranother 3 hours to induce expression. The product expressed was analyzedby SDS-PAGE. After the electrophoresis one half was strained byCoomassie brilliant blue. A dense band was observed at a molecularweight of about 15.5 kD in the lane of the lysate of induced bacterialcells, which accounted for more than 50% of the total proteins of thebacterial cells as judged by scanning. The other half was placed on acasein gel plate after SDS was removed, and incubated at 37° C. forseveral hours. There was a clear region corresponding to 15.5 kD. Inother words, casein at this position was degraded, suggesting thatKGD-Sak had fibrolytic activity. After the cells were crushed andcentrifuged, it was discovered that the 15.5 kD band was mainly presentin the pellet, while it could hardly be observed in the supernatantindicating that the product expressed exists as inclusion bodies.

d. The Inducible Expression in the Engineered Strain

The engineered strains were screened for high level of expression (e.g.the recombinant protein expressed accounted for more than 50% of thetotal protein of the cell). Low density fermentation was carried outwith the strain selected in a 10 L fermentor. After 3 hours oftemperature induction culturing, cells were spun down, washed in PBS,and stored at −70° C. until use. 80 g wet cells were obtained from a 10L culture. The wet cells were suspended in PB buffer, disrupted by ahigh pressure homogenizer, and centrifuged. Samples were taken forSDS-PAGE. The result indicated that the protein of interest was presentin the lane of the pellet with a band stained densely at the position ofa molecular weight of 15.5 kD, and that hardly any stain could beobserved at the corresponding position of the supernatant, suggesting,that RGD-Sak mainly exists as inclusion bodies.

e. Isolation, Solubilization and Renaturation of Inclusion Bodies

After disruption by pressing, 80 g cells of the engineered strain werecentrifuged at 10,000 rpm and 20 g inclusion bodies was obtained. Afterthe inclusion bodies was washed in 0.05 mol/L PB, pH5.2 and centrifugedat 5,000 rpm, it was dissolved in a solution containing 0.1 mol/L PB pH5.2, 6 mol/L guanidium, hydrochloride, 0.5% β-mercaptoethanol, andincubated at room temperature until the solution became clear. Afterultracentrifugation at 30,000 rpm, the pellet was discarded and thesupernatant was diluted for renaturation in 0.1 mol/L PB pH5.0 and 0.5%β-mercaptoethanol.

f. Sephadex G-10 and S-Sepharose FF Column Chromatography

After concentration by ultrafiltration (MW 1000, Millipore), thesupernatant was filtrated through a Sephadex G-10 column. The filtratewas applied to an S-Sepharose FF column equilibrated previously by 10bed volumes of 0.1 mol/L PB buffer pH5.0. A chromatograph (Waters) wasused to control the flow rate and to detect the protein peak. Afterloading, the column was washed to baseline with PB buffer and elutedwith a 0-1 mol/L gradient of NaCl. The fractions eluted, were collected.The distribution of the desired protein was analyzed by SDS-PAGE and theconcentration determined by Bradford method (the reagents used werepurchased from Bio-Rad).

All chromatography operations were of routine work to those skilled inthe art.

g. The Identification of the Purity and the Determination of theMolecular Weight

The sample was analyzed by SDS-PAGE according to Molecular Cloning, ALaboratory Manual. After strained with Coomassie brilliant blue R-250,the gel was scanned with Pharmacia Imagemaster VDS to determine thepurity and molecular weight of the protein. Consequently, it wasdetermined that the purity was above 95% and that the molecular weightwas about 15.5 kD.

h. The Determination of the Biological Activity

Casein gel plaque method (Pipemo A G et al, J. Exp. Med. 48(1) 223-234(1978)) and chromogenic substrate method (Lijnen H R et al, J. Biol.Chem. 266, 11826-11832 (1991)) were carried out to determine thebiological activity. The specific activity was about 90,000-100,000HU/mg. For the definition of the unit, see Tang Q-Q et al, DrugBiotechnology (Chinese) 4(1), 1-4(1997).

i. Determination of the Km and Kcat Value of Sak Plasmin Complex andKGD-Sak Plasmin Complex

2 μmol/L Sak or RGD-Sak was incubated with 2 μmol/L plasminogenrespectively in 0.1 mol/L PB at pH7.4 and 37° C. for 30 min to formcomplexes with plasmin. Then catalytic amount of complex was taken (5nM) to react for 0-10 min in the following systems in 0.1M PB at pH7.4and 37° C., and OD405 was recorded every 30 sec. Each concentration ofplasminogen was assayed in triplicates and averaged.

Reaction System Final Concentration Sak · plasmin (KGD-Sak · plasmin) 5nmol/L chromogenic substrate S-2390 1 mmol/L plasminogen 1-30 μmol/L

The activation of plasminogen by KGD-Sak plasmin corresponded to theMichaelis-Menten equation (table 2).

TABLE 2 the comparison of enzymatic kinetic constants of the plasminogenactivation by KGD-Sak · plasmin and Sak · plasmin Km(μmol · L⁻¹)Kcat(s⁻¹) Kcat/Km Sak · plasmin 6.51 1.06 0.16 RGD-Sak · plasmin 14.101.46 0.10j. Test of Polymerizing Ability

Wild-type Sak was used as a control. The samples were dissolved inphysiological saline. Two protein concentrations 30 mg/ml (high) and 3mg/ml (low), were tested. The solutions were kept at room temperature.Samples were taken every 24 hr and analyzed by electrophoresis.

At both protein concentrations, the polymerizing ability of KGD-Sak wassignificantly lower than that of wild-type Sak.

k. The Sensitizing Test on Guinea Pigs

Both recombinant wild-type Sak and mutant KGD-Sak were dissolved insterile physiological saline at a concentration of 2500 U/ml for thesensitizing test. For each administration, intact vials were taken toprepare the fresh solutions in a sterile way 20 healthy guinea pigs wereassigned to two groups randomly, with 10 guinea pigs each. The guineapigs were i.p. injected with r-Sak or KGD-Sak at a dose of 0.15 mg/kgevery other day for three times. A first and a second i.v. attack at 0.3mg/kg were performed on day 14 and 21, respectively. 2 healthy andnon-injected guinea pigs were i.v. injected with above samples at 0.3mg/kg and observed for the presence of similar response to exclude thepharmacological and pathological interference of the samples.

The group injected with wild-type r-Sak: 8 guinea pigs showed a positiveresponse of grade IV and 2 showed a positive response of grade II.

The group injected with KGD-Sak: 1 guinea pig showed a positive responseof grade I and the others showed no obvious response.

Grade I response: mild cough Grade II response: cough several times,quiver Grade III response: quiver violently Grade IV response:convulsion, spasm, incontinence of the feces and urine, shock to death

The antibody levels in the sera from the guinea pigs immunized for 1-3weeks were tested by ELISA, wherein wt-Sak and KGD-Sak were used asantigens, respectively. In the first week the antibodies against eitherantigen were low. In the second week, the antibody level of the wt-Sakgroup (n=10) increased to 1:800 whereas the antibody lever of theKGD-Sak group (n=10) was 1:200. In the third week, the wt-Sak groupincreased to 1:3200, whereas the RGD-Sak group increased to 1:400. Thus,the immunogenicity of the KGD-Sak decreased significantly as comparedwith wt-Sak.

The above results indicated that the immunogenicity of the KGD-Sak wasdecreased significantly as compared with wt-Sak.

1. The Platelet Aggregation Inhibitory Assay

Fresh blood anticoagulated with 1/10 volume of 110 mmol/L sodium citratewas centrifuged slowly (150 g, 10 min) to get the platelet-rich plasma(HRP). RGD-Sak was added to HRP to a final concentration of 2 μmol/L andthe mixture was incubated at 37° ° C. for 2 min with continuousstirring. Then ADP was added to a final concentration of 2 μmol/L as aninducer. The platelet aggregation rate was determined within 5 min witha two-channel platelet aggregator (CHRONO-LOG 560). Wild-type r-Sak (2μmol/L) and physiological saltine were assayed as controls. ADP waspurchased from Sigma and other reagents were of analytic grade, made inChina.

Consequently, the aggregation rate of the KGD-Sak group (3.8%±1.5%, n=3)was significantly lower than that of the r-Sak group (64%±4%, n=3) andthat of the physiological saline group (60%±3%, n=3), suggesting thatKGD-Sak has a powerful potency to inhibit platelet aggregation inducedby ADP.

m. The Thrombolytic Assay on Animals

The animal thrombolytic assay was performed with KGD-Sak prepared in thepresent invention, verifying that KGD-Sak retained the same thrombolyticproperty as that Of wild-type Sak.

Treating experimental rabbit femoral artery thrombosis with KGD-Sak: thetreatment group of KGD-Sak, the treatment group of wild-type Sak and thecontrol group of blank each consisted of 6 animals. It was indicated byarteriography that the femoral artery under the middle segment was notvisible before treatment. When photography was repeated 60 minutes afteri.v. injection of 0.1 mg/kg KGD-Sak, the femoral artery was filledthoroughly and the blood cycle was recovered which was consistent withthe wild-type Sak group, while in the control group, the femoral arterydid not appear to be filled.

Treating experimental rabbit hyphema with KGD-Sak the treatment group ofKGD-Sak, the treatment group of wild-type Sak and the control group ofblank each consisted of 6 animals 4 hours after intraocular injection of10-20 μg KGD-Sak, it was observed that the hyphema clot was lysed andthe red blood cells settled and formed an interface with aqueous humor.The intraocular hematocele was eliminated after 24 hours. This isconsistent with the wild-type Sak group. However, the hyphema in thecontrol group was not significantly changed.

KGD-Sak thrombolytic therapy was safe and efficient for induced by thetreatment of acute myocardial infraction experimental dog coronaryarterial thrombosis. The experimental group consisting of 6 animals wasgiven KGD-Sak at 0.3 mg/kg body weight by i.v. infusion; and the controlgroup consisting of 6 animals was given physiological saline instead ofKGD-Sak by i.v. infusion. Coronary arteriography was carried out beforeand after dosing Before administration, it was shown that the leftanterior descending branch of the coronary artery was unfilled or filledincompletely in the animals of both groups. Arteriography was performed30 minutes after treatment. It was shown that the left anteriordescending branch was refilled in the animals of the experimental group,and the animals survived. As for the control group, there was nosignificant change in the region filled incompletely and the animalsdied several hours later.

KGD-Sak thrombolytic therapy was safe and efficient the treatment ofacute cerebral infarction induced by experimental pig intracranialarterbial thrombosis. The experimental group Consisting of 6 pigs wasgiven KGD-Sak at 0.2 mg/kg body weight by i.v. infusion; and the controlgroup consisting of 6 pigs was given physiological saline instead ofKGD-Sak by i.v. infusion. Intracranial DSA arteriography was performedbefore and after administration. Before administration, it was shownthat there were some intracranial arteries filled incompletely in theanimal of both groups. Angiography was performed 30 minutes aftertreatment. It was shown that the intracranial arteries were refilled inthe animals of the experimental group, and the animals survived. As forthe control group, there was no significant change in the intracranialarteries filled incompletely and the animals died several days later.

Without further detailed description, those skilled in the art can applythe invention to the maximum in the light of the foregoing teaching.Thus, it is to be understood that the preferred specific embodimentsabove are intended as illustrations, but in no way to limit the scope ofthe invention.

All the references cited herein are incorporated by reference in theirentireties.

The substantial characteristics of the invention will become apparent tothose skilled in the art from the foregoing description. Moreover,various modifications and improvement of the invention may be madewithout departing from the spirit of the present, inventions. Suchmodifications and improvements are intended to fall within the scope ofthe appended claims.

1. A recombinant staphylokinase derivative with bifunctionality of athrombolytic and an anticoagulant, having the amino acid sequence of SEQID NO: 1 or SEQ ID NO:
 3. 2. The recombinant staphylokinase derivativeof claim 1, characterized in that it comprises a sequence that has 15amino acids deleted beginning at the NH₂-terminal of SEQ ID NO: 1 or SEQID NO: 3, with Ser at residue 16 being substituted by Lys.
 3. Therecombinant staphylokinase derivative of claim 1 comprising the aminoacid sequence of SEQ ID NO:
 1. 4. The recombinant staphylokinasederivative of claim 1 comprising the amino acid sequence of SEQ ID NO:3.